The Plant Kingdom is divided into two phyla, the Bryophyta and Tracheophyta. The phylum Tracheophyta includes over 266,000 species grouped into four subphyla. Subphylum pteropsida includes Class Angiospermae. This class is divided into two subclasses, the dicotyledons (dicots) and the monocotyledons (monocots).
Since monocots include many of the important grain and feed crops, plant geneticists are keenly interested in being able to produce transgenic monocots. About 50,000 species of monocots are known. These include the lilies, palms, orchids, irises, tulips, sedges, and grasses. The grasses include corn, wheat, rice and all other cereal grains. Unfortunately, monocots have been extremely difficult to genetically engineer so that most of the work with plants has been with dicots.
The dicots are the larger of the two groups, with approximately 200,000 species known. The buttercup, snapdragon, carnation, magnolia, poppy, cabbage, rose, pea, poinsettia, cotton plant, cactus, carrot, blueberry, mint, tomato, sunflower, elm, oak, and maple represent 19 of the 250 families of dicots.
The genetic information within a DNA molecule usually serves as the template for the synthesis of a large number of shorter RNA molecules, most of which in turn serve as templates for the synthesis of specific polypeptide chains. Specific nucleotide segments, (often called promoters), are recognized by RNA polymerase molecules that start RNA synthesis. After transcription of a functional RNA chain is finished, a second class of signals leads to the termination of RNA synthesis and the detachment of RNA polymerase molecules from their respective DNA templates.
Currently, there are a number of common promoters used to drive heterologous gene expression in monocot plants.
David McElroy, et al., (1990) noted that transient expression assays of a construct in which the promoter from the rice actin 1 gene (Act 1) was fused to the bacterial .beta.-glucuronidase gene (GUS) in transformed rice protoplasts showed that the actin promoter drives high levels of gene expression. This expression was about 6-fold greater than that seen with the maize alcohol dehydrogenase 1 gene (Adh1) promoter, and is dependent on the presence of an intact Act1 5' intron.
David McElroy, et al., (1991) noted that optimized vectors for monocot transformation were constructed using either the Cauliflower Mosaic Virus (CaMV) 35S promoter or the Act1 promoter. Transient expression assays were done on both transformed rice and maize protoplasts. Addition of the Act1 intron and optimized GUS translation initiation site to either promoter sequence increased gene expression significantly. It is also noted as an unpublished result that the actin promoter was shown to drive GUS expression in transient assays of wheat, oat, barley and sorghum protoplasts.
Wanggen Zhang, et al., (1991) noted that in situ hybridization studies of transgenic rice plants carrying an Act1-GUS gene fusion showed that the Act1 promoter has a constitutive pattern of expression in both vegetative and reproductive tissue.
Jun Cao, et al., (1992) noted that transgenic rice plants were selected on bialophos following transformation with the bar gene expressed under the control of either the CaMV 355 promoter or the rice Act1 promoter.
Alan H. Christensen, et al., (1992) noted that Maize protoplasts transformed with a maize Ubi-1-CAT gene fusion showed approximately 10-fold higher levels of CAT activity than maize cells transformed with a CaMV-35S-GUS gene fusion in transient expression assays. Northern blot analysis of Ubi-1 and Ubi-2 transcript levels following heat shock of maize seedlings demonstrated that both genes are expressed constitutively at 25C, but are induced following the heat shock.
Seiichi Toki, et al., (1992) noted that stably transformed transgenic rice plants were obtained following electroporation-mediated transformation of the bar gene expressed under the control of the maize Ubi-1 promoter and selection on bialophos. This result demonstrates that the Ubi-1 promoter can be used to drive sufficiently high levels of gene expression in rice to allow for selection and regeneration of fertile, transgenic rice plants.
J. Troy, et al., (1993) noted that stably transformed wheat plants were obtained following bombardment of calli derived from immature embryos with both the bar gene and GUS, each being expressed under the control of the maize Ubi-1 promoter, followed by selection on bialophos. This result demonstrates that the Ubi-1 promoter can be used to drive sufficiently high levels of gene expression in wheat to allow for selection and regeneration of fertile, transgenic plants.
Yuechun Wan, and Peggy G. Lemaux, (1994) noted that stably transformed, fertile barley plants were obtained after microprojectile bombardment of embryonic barley tissues with both the bar gene and GUS, each being expressed under the control of the maize Ubi-1 promoter, followed by selection on bialophos. This result demonstrates that the Ubi-1 promoter can be used to drive sufficiently high levels of gene expression in barley to allow for selection and regeneration of fertile, transgenic plants. An experiment involving bombardment of a small number of plants with either Ubi-bar or CAXV 35S-bar showed no significant difference in the number of transformants obtained.
Junko Kyozuka, et al., (1991) noted that a maize Alh1 promoter-GUS fusion was introduced into rice protoplasts to obtain transgenic rice plants. The GUS activity in the transgenic plants was examined to determine the pattern of GUS expression. The maize Adh1 promoter was found to promote constitutive expression in all parts of the plants examined. As had been previously demonstrated for Adh1 expression in maize, the Adh1-driven GUS expression was induced in roots by anaerobic conditions.
D. I. Last, et al., (1991) noted that when the maize Adh1 promoter was modified by addition of multiple copies of the Anaerobic Responsive element of the maize ADH1 gene and ocs elements from the octopine synthase gene of Agrobacterium tumefaciens (pEmu). In transient expression assays of protoplasts of different moncot species transformed with pEmu-GUS, the best construct, gave 10-50-fold higher levels of expression than the CaMV 35S promoter in wheat, maize, rice, einkorn, and lolium multiflorum.
Robert Bower and Robert G. Birch (1992) noted that stable transformants were obtained following transformation of embryogenic callus of sugarcane with the neomycin phosphotransferase gene under the control of the Emu promoter.
D. A. Chamberlain, et al., (1994) noted that the Emu promoter was used to drive the expression of four different selectable marker genes (neomycin phosphotransferase, hygromycin phosphotransferase, phophinothryicin N-acetyltransferase and a mutant acetolactate synthase conferring herbicide resistance) that were transformed into both wheat and rice. Wheat callus and transformed rice plants were obtained after selection of transformants, demonstrating that the promoter can be used to drive expression of selectable marker genes to obtain transformed cereals.
A review of promoter elements used to control foreign gene expression in transgenic cereals has recently been published and is herein incorporated by reference (McElroy and Brettel, Tibtech, Vol. 12, February, 1994).
A number of promoters are currently being used for transformation of dicotyledonous plants. These promoters come from a variety of different sources. One group of commonly used promoters were isolated from Agrobacterium tumefaciens, where they function to drive the expression of opine synthase genes carried on the T-DNA segment that is integrated into the plant genome during infection. These promoters include the octopine synthase (ocs) promoter (L. Comai et al., 1985; C. Waldron et al., 1985), the mannopine synthase (mas) promoter (L. Comai et al., 1985; K. E. McBride and K. R. Summerfelt, 1990) and the nopaline synthase (nos) promoter (M. W. Bevan et al., 1983; L. Herrera-Estrella et al., 1983, R. T. Fraley et al., 1983, M. De Block et al., 1984;, R. Hain et al., 1985). These promoters are active in a wide variety of plant tissue.
Several viral promoters are also used to, drive heterologous gene expression in dicots (J. C. Kridl and R. M. Goodman, 1986). The Cauliflower Mosaic Virus 35S promoter is one of the promoters used most often for dicot transformation because it confers high levels of gene expression in almost all tissues (J. Odell et al., 1985; D. W. Ow et al., 1986; D. M. Shah et al., 1986). Modifications of this promoter are also used, including a configuration with two tandem 35S promoters (R. Kay et al.,1987) and the mas-35S promoter (L. Comai et al., 1990), which consists of the mannopine synthase promoter in tandem with the 35S promoter. Both of these promoters drive even higher levels of gene expression than a single copy of the 35S promoter. Other viral promoters that have been used include the Cauliflower Mosaic Virus 19S promoter (J. Paszkowski et al., 1984; E. Balazs et al.) and the 34S promoter from the figwort mosaic virus (M. Sanger et al., 1990).
Studies of AHAS expression in a number of plants indicates that AHAS is expressed in all plant tissues. Gail Schmitt and Bijay K. Singh (1990) noted that enzyme assays performed on various tissues of lima bean demonstrated that AHAS activity was present in all tissues tested, including leaves, stems, roots, flowers, pods and meristems. AHAS activity was found to be fairly constant in the stems, but declined in leaves, roots and meristems with increasing age.
Sharon J. Keeler et al., (1993) noted that tobacco contains two genes encoding acetohydroxyacid synthase, SurA and SurB. Both genes appear to be expressed in all tissue types, with about a four-fold variation in the level of expression in different tissues. Developing organs appear to have the highest levels of expression. In situ hybridization studies demonstrated that the highest levels of expression were consistently observed in metabolically active or rapidly dividing cells. SurB was expressed at higher levels than SurA in all tissues examined.
Therese Ouellet et al., (1992) noted that Brassica species contain multigene families encoding acetohydroxyacid synthase. Four of the five AHAS genes have been identified in Brassica napus. RNAse protection assays using gene-specific probes were performed to determine the patterns of expression of the different members of the gene family in various Brassica species. Two of the genes, AHAS1 and AHAS3, were found to be expressed constitutively in all tissues examined. AHAS2 transcripts were detected only in the reproductive organs and extra-embryonic tissue of seeds. Transcripts encoded by the fourth gene, AHAS4, were not detected and it is therefore believed that this represents a pseudogene.
Dale L. Shaner and N. Moorthy Mallipudi (1991) noted that comparison of AHAS activity in young corn leaves and BMS cells grown in suspension culture showed that the activity of the BMS cells per gram fresh weight was approximately 5.8-fold higher than in the leaf samples. Since the BMS cells are actively dividing, this result is consistent with results of previous studies with tobacco and lima bean which demonstrated that younger, actively dividing tissues have more AHAS activity than older tissues.